CN101824390B - RY2 strain of root nodule nitrogen-fixing bacterial strain system and application thereof - Google Patents
RY2 strain of root nodule nitrogen-fixing bacterial strain system and application thereof Download PDFInfo
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- CN101824390B CN101824390B CN2009102143155A CN200910214315A CN101824390B CN 101824390 B CN101824390 B CN 101824390B CN 2009102143155 A CN2009102143155 A CN 2009102143155A CN 200910214315 A CN200910214315 A CN 200910214315A CN 101824390 B CN101824390 B CN 101824390B
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Abstract
The invention discloses a RY2 strain of a root nodule nitrogen-fixing bacterial strain system and application thereof. The strain (Bradyrhizobium yuanmingense RY2) is preserved in the China Center for Type Culture Collection (CCTCC) on 13th November in 2009. The preservation number is CCTCC NO:M 209266. The RY2 strain of the root module nitrogen-fixing bacterial strain system can exclusively greatly improve the nitrogenase activity of TPRC2, so that the nitrogenase activity of the TPRC2 is over three times higher than that of TPRC5 and can achieve higher nitrogenase activity by using a few root nodules. The yield of stylosanthes guianensis can be improved by over 30 to 40 percent by inoculating the root nodule bacteria, and the root nodule bacteria can be suitable for intercropping of stylosanthes guianensis TPRC2 and provides a large number of nitrogen for the stylosanthes guianensis, crops and soil. Meanwhile, the RY2 has strong salt tolerance, can grow in the environment that the NaCl concentration is higher than 0.25mol/L, can be planted in the salinized soil and has better effects of improving the yield of the stylosanthes guianensis and utilizing and improving the salinized soil.
Description
Technical field
The invention belongs to plant nutrition and forage cultivating technical field, be specifically related to a kind of biological nitrogen fixation technology, especially relate to a kind of root nodule bacterium that can make the efficient dross of khuskhus and reach high yield.
Background technology
Khuskhus (Stylosanthes guianensis SW.) has another name called tropical clover, Brazilian clover, originates in South America.Nineteen eighty-two is drawn from CIAT (CIAT) by Chinese Academy of Tropical Agricultural Sciences, and plants experimentally successfully in Hainan, and big area is extended to each province, south China (district) earlier.Khuskhus contains crude protein 16.0～20.0% in the dry-matter of full-bloom stage, crude fat 1.8～2.5% is rented fiber 17.0～19.0%, nitrogen-free extract 38.0～44.0%, and coarse ash 8.0～10.0% is a kind of good tropical leguminous forage.(district) economized in southern china such as Guangdong, Guangxi etc., with planting between khuskhus and fruit tree, except being used for forage grass, also can play covering, conserve water and soil and the green manure effect.If with mixed seeding such as graminous pasture such as Herba Setariae Viridis, can build up good artificial pasture and be used to herd.
Khuskhus selects usually that draining is good, soil layer is deep, the husky preferably earth of soil property or loam plantation.But do not form root nodule at lean soil sowing khuskhus, or only have some to infect the ineffetive nodulation that forms by indigenous root nodule, the legume inoculation root nodule bacterium can increase increment 15～50% on the barren soil.For improving khuskhus dross rate, the fixed nitrogen loam should be used the seed dressing of khuskhus root nodule bacterium, can effectively improve the dross rate of khuskhus.Therefore the screening root nodule bacterium that can make the efficient dross of khuskhus and reach high yield have great importance.
Khuskhus with the characteristic of its high yield, high-quality and anti-low level management after introduction, become me very soon China south leguminous forage promote mainly kind.The content of khuskhus original inhabitants bacterium is extremely low at the beginning of khuskhus is introduced because in the planting site soil, and khuskhus is nodulation and nitrogen fixation well, and main dependence fertilising obtains high yield.So just can not embody real high yield and anti-low level management.China had introduced Rhizobium Inoculant from Australia and had been applied to the khuskhus planting site afterwards, and the output of khuskhus is improved.But because Australian Rhizobium Inoculant is based on the efficient bacterial strain that Australian soil and climatope are selected, just can a spot of raising khuskhus output to the weather of China and edatope, can not reach the such high yield effect of Australia.At present China does not also filter out the bacterial strain that is suitable for China's weather edaphic condition, so screen the khuskhus high-efficiency nitrogen-fixing root nodule bacterium that are suitable for the specific region from the indigenous bacterium of the Different Soil conditions of China column with cultivations of flower or grass flowers and plants important practical sense is arranged.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, it is RY2 that a kind of nodule azotobacter strain is provided, a kind of root nodule bacterium that can make the efficient dross of khuskhus and reach high yield.
The inventor November 14 in 2008 in Yunnan Agriculatural Academy's heat through institute experimental field in (N=24 ° 45.684 ', E=98 ° 55.546 ', H=1023m) collect the root nodule of the khuskhus of the many riotous growth of a strain dross, cut the khuskhus over-ground part whole root system is placed on cryopreservation in the ice bag together with the root nodule freshness protection package of packing into, through separate and purifying to obtain a kind of nodule azotobacter strain be RY2 (Bradyrhizobium yuanmingense RY2), be preserved in Chinese typical culture collection center on November 13rd, 2009, preserving number is CCTCC No:M 209266.Its 16sDNA sequence is shown in SEQ ID NO 1.
Another object of the present invention provides the cultural method that described nodule azotobacter strain is RY2.
The present invention is the root nodule that collects, cut the khuskhus over-ground part with whole root system together with root nodule pack into freshness protection package be placed on the follow-up preservation back-up of low temperature in the ice bag from and purifying.
Concrete separation and purge process:
Take out the root system of khuskhus and rinse well from ice bag, transfer in the culture dish after the sterilization, root nodule is smashed in sterilization to pieces, dips in to get oyster white juice and rule on ready YMA solid medium.Be inverted in and place incubator (28 ± 1 ℃, dark) cultivation in the freshness protection package drawing a good plate.Note checking on the substratum whether grow bacterium colony in the culturing process, the mark bacterium colony, record form and glossiness are defined as root nodule bacterium.
Get rid of behind the non-root nodule bacterium bacterium colony according to the bacterium colony of above-mentioned mark will grow the time phase difference of single bacterium colony more than 3 days bacterium colony and plate on growthhabit, bacterium colony size, bacterium colony transparency, and the light inconsistent bacterium colony of refraction, colony colour down choose, on new YMA medium, draw plate and number.Each time to the new bacterium colony of drawing plate all adopt aforesaid method carry out purifying until the colony growth form on the same block of plate, bacterium colony size, bacterium colony transparency, and light under refraction, colony colour unanimity till.Undertaken putting under in the YMA inclined-plane behind microscopy and the tieback by the plate after with purifying of purifying step by step and preserve.
Described colonial morphology: the translucent or White-opalescent point-like thing of root nodule bacterium early growth period bacterium colony water sample, later stage bacterium colony be circle, oyster white, translucent, neat in edge, cement more or less.Cultivate 2～4 days colony diameters and reach 2～4mm and give birth to the type root nodule bacterium for fast, cultivate 5～10 days bacterium colony colony diameters just 1mm be living type root nodule bacterium slowly.
B. thalli morphology: mark is tentatively confirmed as the root nodule bacterium bacterium colony, and therefrom picking lawn smear carries out gramstaining, observes under 100 power microscopes.Root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain beta-hydroxy-butanoic acid, promptly reflect strong like the cavity particle or make thalline in the form of link, Gram-negative (G-), no gemma, thalline is single or paired.
It is the microbial inoculum that RY2 prepares that a further object of the invention provides described nodule azotobacter strain.Shake the bacteria suspension that bacterium obtains in the liquid YMA medium (bacteria containing amount is greater than 1.0 * 10 by the bacterium on the flat board is washed
9Individual) seed dressing, directly tieback is to plant root or mix soil.For example the peat composed of rotten mosses, vermiculite or perlite etc. are made microbial inoculum use, the preferred 10ml bacterium liquid of the blending ratio/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite also can to mix the thalline sorbing material.。
A further object of the invention provides khuskhus and inoculates the method that described nodule azotobacter strain is the RY2 bacterium.To be the RY2 bacterium prepare liquid bacterial agent and can be prior to seeding soak the plant root with the seed soaking of bacterium liquid, before growing seedlings, water in the khuskhus root soil after transplanting by shaking bacterium described nodule azotobacter strain, also can be with bacterium liquid mixings thalline sorbing material mixed imposing in the soil before for example the peat composed of rotten mosses, vermiculite, perlite etc. are made microbial inoculum and transplanted.
The present invention gets beneficial effect:
Behind the TPRC2 khuskhus, the nitrogenase activity of khuskhus is greatly improved the RY2 tieback.Making microbial inoculum, to be applied to behind the soil raising to TPRC2 output be a times of TPRC5, will make the khuskhus hay yield increase more than 3 times if big area is applied to the big Tanaka of single cropping TPRC2; RY2 still is a rhizobium strains that salt tolerance is more intense, the fine growth of RY2 energy under the salt concn that other bacterium can not grow, and this provides technical guarantee to improving khuskhus output in China's south salt-affected soil.
Description of drawings
Fig. 1 root nodule bacterium RY2 tieback check figure
Fig. 2 root nodule bacterium RY2 bacterium colony figure
Fig. 3 root nodule bacterium RY2 gramstaining picture
Dross situation behind Fig. 4 tieback root fungus RY2
Fig. 5 RY2 agarose gel electrophoresis figure
Fig. 6 NCBI comparison chart
Fig. 7 RY2 root nodule bacterium cluster analysis figure
Fig. 8 tieback RY2TPRC2, TPRC5 change of production figure
Fig. 9 RY2 aciduric bacteria liquid OD value
The anti-NaCl bacterium of Figure 10 RY2 liquid OD value
Figure 11 tieback RY2 is to khuskhus yield effect figure
Embodiment
Further describe the present invention below in conjunction with specific embodiments and the drawings.
Acquisition and the cultivation of embodiment 1 root nodule bacterium RY2
November 14 in 2008 in Yunnan Agriculatural Academy's heat through institute experimental field in (N=24 ° 45.684 ', E=98 ° 55.546 ', H=1023m) collect the root nodule of the khuskhus of the many riotous growth of a strain dross, cut the khuskhus over-ground part whole root system is placed on cryopreservation in the ice bag together with the root nodule freshness protection package of packing into.Through separate and purifying to obtain a kind of nodule azotobacter strain be RY2 (Bradyrhizobium yuanmingense RY2), be preserved in Chinese typical culture collection center on November 13rd, 2009, preserving number is CCTCC NO:M 209266.
(1) root system that takes out khuskhus from ice bag washes under tap water and 2～3 times root system is rinsed well, cuts in culture dish fresh root complete, root nodule band 2mm that color is dark red with 2 grades of water that surface washing is clean with scissors; This process will be guaranteed the complete of root nodule surface.
(2) root nodule of rinsing well is transferred in the culture dish after the sterilization, add alcohol-pickled 3 minutes of 95% (volume by volume concentration) after, alcohol is poured out, with aseptic water washing 4～5 times, add 0.1% (quality volumetric concentration, 0.1g HgCl
2/ 100ml water) HgCl
2Solution sterilization 2～3 minutes is rapidly with HgCl
2Pour out and add sterilized water, shift the Bechtop operation, with aseptic water washing more than 6 times, sterilized water is poured out, choose in the culture dish after root nodule is transferred to sterilization and (can on flame, sterilize), with after the sterilization tweezers and transfering loop root nodule is smashed to pieces, dip in and get oyster white juice and on ready YMA solid medium, rule.Be inverted in and place incubator (28 ℃, dark) cultivation in the freshness protection package drawing a good plate.
The YMA medium prescription is as table 1, and regulating pH value is 6.
Table 1YMA (Yeast Mannitol Agar) culture medium prescription (L
-1)
(3) began to check whether grow bacterium colony on the substratum at the 3rd day that cultivates, until about the 15th day, the bacterium colony mark is come out to write down form and glossiness.
In culturing process, determine whether to be root nodule bacterium from following 3:
A. colonial morphology: the translucent or White-opalescent point-like thing of root nodule bacterium early growth period bacterium colony water sample, later stage bacterium colony be circle, oyster white, translucent, neat in edge, cement more or less.Cultivate 2～4 days colony diameters and reach 2～4mm and give birth to the type root nodule bacterium for fast, cultivate 5～10 days bacterium colony colony diameters just 1mm be living type root nodule bacterium slowly.
B. thalli morphology: mark is tentatively confirmed as the root nodule bacterium bacterium colony, and therefrom picking lawn smear carries out gramstaining, observes under 100 power microscopes.Root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain beta-hydroxy-butanoic acid, promptly reflect strong like the cavity particle or make thalline in the form of link, Gram-negative (G-), no gemma, thalline is single or paired;
C. by isolated root nodule bacterium are received on the khuskhus next time at aseptic condition, can dross then be root nodule bacterium.
The morphological specificity of root nodule bacterium RY2:
Root nodule bacterium RY2 is a kind of of knurl Pseudomonas of taking root slowly, cultivate the vestige that is coated with along the first stroke after 50 hours and begin to have the banded translucent projection of water sample, began obviously and independently became gradually independent bacterium colony by the 4th day with rearward projection, the bacterium colony circle of 1.0～2.0mm appears in finishing touch after the 5th day, the edge is complete, smooth surface, protruding globule shape, the water white transparency degree is high, and viscous secretion is less.See the bacterium colony of RY2 root nodule bacterium shown in the accompanying drawing 2.
RY2 number optimum growing condition is: 28 ℃ of temperature, pH value are 6, rotating speed 180r/min.It is former former with nitrogen to use carbon widely, can well grow on the comprehensive extract of various plant origins, better than growth on protein culture medium on the YMA medium.
The physiological property of root nodule bacterium RY2
Root nodule bacterium RY2 is purple through gramstaining at microscopically, is shaped as little rod-short body, and size is 0.5～0.9 μ m * 1.2～3.0 μ m, and cell is single or paired, and Chang Kejian arranges in groups.Be Gram-negative bacteria.See the gramstaining of root nodule bacterium RY2 shown in the accompanying drawing 3 picture.
(4) get rid of non-root nodule bacterium bacterium colony, according to the bacterium colony of above-mentioned mark will grow the time phase difference of single bacterium colony more than 3 days bacterium colony and plate on growthhabit, bacterium colony size, bacterium colony transparency, and the light inconsistent bacterium colony of refraction, colony colour down choose, on new substratum, draw plate and number.Each time to the new bacterium colony of drawing plate all adopt aforesaid method carry out purifying until the colony growth form on the same block of plate, bacterium colony size, bacterium colony transparency, and light under refraction, colony colour unanimity till.Undertaken putting under in the YMA inclined-plane behind microscopy and the tieback by the plate after with purifying of purifying step by step and preserve.
(5) tieback checking khuskhus root nodule bacterium
A. the preparation of bacterium liquid
The root nodule RY2 that separation and purification is come out washes in the ready liquid YMA medium with sterilized water, seals with sealing film, and 180r/min shakes bacterium is measured bacterium liquid after 3 days OD value in shaking table, and (every ml bacterium liquid bacteria containing amount is more than 1.0 * 10 greater than 0.6 the time as OD
9The individual bacterium liquid that promptly gets can be used in tieback.Described liquid YMA medium is in the substratum of table 1 agar to be removed, and the adjusting pH value is 7.
B. the preparation of aseptic seedling
Selected khuskhus seed number grain soaked 5 minutes in 95% ethanol, took out the back at 0.2% HgCl
2In handled 5 minutes, with aseptic water washing 5～10 times, come the bacterium of going out then, put well in the culture dish of germination paper, soak germination paper with the 0.05mmol/L calcium sulphate soln of the bacterium of going out, be placed in 28 ℃ the incubator dark vernalization 3 days.When treating seedling length to 4 centimetre seedling is moved in the sand training case of the ready bacterium of going out, stand-by.
C. tieback
From sand training case, choose sturdy seedling and be put into the bacterium liquid immersion for preparing with step (5) a in the culture dish 15 minutes, with tweezers seedling is planted in the small flower of filling sterilization sand, the shape that assumes diamond in shape in every basin 4 strains of planting, every seedling adds bacterium liquid 1～2ml, guarantees that the bacterium amount that connects of every naked seedling reaches 1.0 * 10
9More than individual.Pull up seedling after approximately and whether check dross, dross then is root nodule bacterium.Root nodule RY2 dross after the tieback check is obvious, and provable isolate is pure root nodule bacterium.See the tieback of root nodule bacterium RY2 shown in the accompanying drawing 1 assay.
The 16S rDNA sequence order-checking of embodiment 2 root nodule bacterium RY2 and determining of classification
In order to determine the phylogeny status of root nodule bacterium RY2, the 16SDNA series of the bacterial strain that obtains checks order to separate.At first utilize the test kit of omega company to carry out the extraction of total DNA, utilize primer to carry out the PCR specific amplification then.
Upstream primer 35fc:CTKAAGAGTTTGATCMTGGCTCAGATTGAAC;
Downstream primer 1492r:TACGGYTACCTTGTTACGACTT.
Reaction conditions is as follows:
The PCR reaction
10×Reaction Buffer 5.0μL
dNTPs(10mM) 1.0μL
P35fc (25Pmol) primer 0.5 μ L
P1492r (25Pmol) primer 0.5 μ L
Taq archaeal dna polymerase (5u/ μ L) 1.0 μ L
Template DNA 1.0 μ L
Mend ultrapure water to 41 μ L
94 ℃ of pre-sex change: 3min
94 ℃ of sex change 50s
35 circulations of 56 ℃ of annealing 50s
72 ℃ are extended 60s
72 ℃ of last 5min that extend
Amplified production assay on 1.0% agarose gel electrophoresis is seen the figure of RY2 agarose gel electrophoresis shown in the accompanying drawing 5, send Beijing AudioCodes biotech company to check order, and sequencing result is shown in SEQID NO 1.
The sequence results of gained is compared at the U.S. U.S. state-run bioinformation center (NCBI), find that rhizobium strains RY2 and known strains A B509380Bradyrhizobium yuanmingense similarity reach 100%, see NCBI comparison chart shown in the accompanying drawing 6.Application software DNAStar and TREECONW carry out cluster to the bacterial strain of being surveyed, and see the cluster analysis of RY2 root nodule bacterium shown in the accompanying drawing 7 figure.By comparison result and dendrogram as can be known RY2 be the new strain system of the knurl Pseudomonas (Bradyrhizobium) that takes root slowly, called after heat is ground (RY2) No. 2.
The application experiment of embodiment 3 root nodule bacterium RY2
In order to confirm the effect of RY2 to khuskhus, adopt two main khuskhus kinds (TPRC2, TPRC5), with the method for sand training with root nodule RY2 tieback to the khuskhus root, each handles 4 repetitions (processing of bacterium, sand and seedling is referring to the tieback experimental procedure), not inoculate root nodule is contrast (CK), carries out the tieback contrast.Adopt low nitrogen nutrition liquid to water in the whole process.Influence sees Table 2 and accompanying drawing 8 to the physical signs of khuskhus behind khuskhus TPRC2, the TPRC5 tieback root nodule bacterium RY2.Left side square column (a) is TPRC5 in the accompanying drawing 8, and the right square column (b) is TPRC2 (other histograms does not have direct relation for existing root nodule bacterium results of comparison with the technology of the present invention in the accompanying drawing 8, do not do mark).
The root nodule number of khuskhus TPRC2, TPRC5, root nodule dry weight, plant height, plant fresh weight and nitrogenase activity all increase considerably than the contrast (CK) of correspondence behind the tieback root nodule bacterium RY2 as can be seen from Table 2, and it is extremely remarkable that difference reaches.Particularly nitrogenase activity is very high.Just so big nitrogenase activity can be arranged with few root nodule like this, can form more root nodule if change cultivation step, RY2 will improve the nitrogenase activity of TPRC5 so, is that plant, soil and intercropping plant are improved a large amount of nitrogen.
Nitrogenase activity is to weigh root nodule bacterium and the plant index in conjunction with the lonely ability size in back, nitrogenase activity is active then represent greatly bacterial strain and corresponding plant in conjunction with after good fixed nitrogen, plant just has very high yield potential.The khuskhus root nodule is the symbiotic specificity system of root nodule bacterium and khuskhus, and the khuskhus of different varieties is also different with the result that collocation showed of different fungus strains.
Table 2 root nodule bacterium RY2 tieback behind TPRC2, the TPRC5 to the influence of khuskhus physical signs
Research summary by experiment, after root nodule bacterium RY2 and the symbiosis of TPRC2 khuskhus, the plant height of TPRC2 khuskhus is improved greatly, more high than TPRC5 simultaneously, the nitrogenase activity value of root nodule also improves greatly after the dross, TPRC2 there is very strong yield potential, for providing good bacterial strain as other plant for nitrogen between TPRC2.See the dross situation behind the accompanying drawing 4 tieback root nodule bacterium RY2.
When thalline is released in the physical environment, harmless to people, animal and plant, do not pollute the environment, increased the colony of root nodule bacterium between soil on the contrary, the nodulation and nitrogen fixation of khuskhus there is promoted effect.
The general pH value of soils in south china is low, and khuskhus adapts to southern acid soil except the acidproof system of himself, and root nodule bacterium have also played the effect of balance acid.Find that in the resistance experiment root nodule bacterium can make the pH of substratum rise, its pH value of fast more bacterial strain of growing rises also big more.After each bacterial strain is activated, be inoculated in the YMA liquid nutrient medium, put 28 ± 1 ℃ of cultivations down in the constant temperature oscillator, treat OD
600Be about 1, drawing 50 μ l bacterial suspension inoculations respectively is 4,4.5,4.8,5,5.5,6,7 (volume is 50ml) to the pH value, put 28 ± 1 ℃ of 180rpm shaking culture in the constant temperature oscillator, detect the growing state of bacterium with the variation of nutrient solution opacity, cultivate and use spectrophotometer measurement OD after 72 hours
600Each handles 3 repetitions.The growing state of RY2 in different PH seen accompanying drawing 9.The RY2 root nodule bacterium are undesired less than growth in 4.8 o'clock at PH as can be seen from accompanying drawing 9, can not form greater than 10
9The bacterium liquid of individual bacterium/ml.Can form normal bacterium liquid when PH is 5, the growth of RY2 reaches best when pH value is 5.5.PH descends a little greater than 6 back growth activities.
Most ofly can make the root nodule bacterium of leguminous plants drosses such as pea and trifolium all very sensitive, and high density NaCl can reduce the number of root nodule bacterium in the leguminous plants inoculum to salt.So the salt tolerance of root nodule bacterium is very important to improving their survival abilities in soil and competitiveness.The experiment of salt tolerant resistance adopts the YMA liquid nutrient medium that adds NaCl to cultivate root nodule bacterium, and with the OD value of spectrophotometric determination bacterium liquid, the size of OD value can reflect the situation of bacteria growing speed after 3 days.After each bacterial strain is activated, be inoculated in the YMA liquid nutrient medium, put 28 ± 1 ℃ of cultivations down in the constant temperature oscillator, treat OD
600Be about 1, draw respectively 50 μ l bacterial suspension inoculations to NaCl concentration be 0,0.05,0.08,0.10,0.15,0.20,0.25, in the 0.30mol/LYMA liquid nutrient medium (pH6.0, volume are 50ml), put 28 ± 1 ℃, 180rpm shaking culture in the constant temperature oscillator, detect the growing state of bacterium with the variation of nutrient solution opacity, cultivate and use spectrophotometer measurement OD after 72 hours
600Each handles 3 repetitions.Growing state such as the accompanying drawing 10 of RY2 in different salt concn, curve RY2 is the salt tolerance curve of root nodule bacterium RY2 in the accompanying drawing 10, other several curves are for having the empirical curve of rhizobium leguminosarum now, and are irrelevant with the technology of the present invention, so do not mark.The salt tolerance of RY2 is very strong as can be seen from accompanying drawing 10, when salt concn can also form 10 during for 0.1mol/L
9Individual bacterium/ml bacterium liquid, and most of the root nodule bacterium of leguminous plants drosses such as pea and trifolium are not grown substantially when concentration is 0.08mol/L.The salt condition to RY2 growth restriction can reach 0.25mol/L.RY2 grows fast not as good as other bacterium when salt-free or low concentration of salt, when the growth of salinity RY2 during greater than 0.05mol/L just surpasses other bacterium.So the RY2 utmost point is applicable in the salt-affected soil that the volume increase of khuskhus and the improvement of salt-affected soil are all had great promoter action.
Described microbial inoculum can after shaking bacterium, dress seed processing, water in the khuskhus root of just having transplanted seedlings with liquid bacterial agent.Mix before also can mixing the thalline sorbing material for example sorbent materials such as the peat composed of rotten mosses, vermiculite or perlite being made microbial inoculum and transplanted and impose in the soil.Described blending ratio is 10ml bacterium liquid/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite.
As above husky culture method is prepared aseptic seed and is grown seedlings.Root nodule bacterium are washed the triangular flask 28 ℃ from solid medium cultivated 96 hours, with sterilized water wash-out thalline, and with the sterilized water dilution, with the whirlpool even thalline of device that turns round and round, measure OD value (go into=600nm), the bacteria containing amount that guarantees every seed when the field is inoculated is greater than 10
9Individual.It is standby at 40 minutes postcooling of 121 ℃ of sterilizations the peat composed of rotten mosses to be pulverized (crossing the 2mm sieve aperture) back.Prior to seeding with different bacterium liquid in 10ml bacterium liquid: the ratio of the 50g peat composed of rotten mosses is poured in the peat composed of rotten mosses, and thorough mixing absorbs bacterium liquid by the peat composed of rotten mosses.The preceding microbial inoculum that mixes is mixed of growing seedlings imposes in the soil.The sub-district area is 20m
2, establish four repetitions.Receive sample after three months, it the results are shown in accompanying drawing 11, and the right square column (a) is TPRC5 in the accompanying drawing 11, and left side square column (b) is TPRC2 (other histograms does not have direct relation for existing root nodule bacterium results of comparison with the technology of the present invention in the accompanying drawing 11, do not do mark).
As seen to be regardless of the output difference little in the ground of TPRC2 and TPRC5 in tieback root nodule bacterium not from accompanying drawing 11.At tieback after the different bacterium output compare all with contrast and increase.Other the difference of bacterium of the output of khuskhus TPRC5 and tieback is little concerning RY2, but the output of khuskhus TPRC2 just has significantly and improves, and is more than 4 times of CK.
SEQUENCE LISTING
＜110〉Liu Guodao, Huang Yanxia, Dong Rongshu
＜120〉a kind of RY 2 strain of root nodule nitrogen-fixing bacterial strain system and application thereof
<130>
<160>1
<170>PatentIn version 3.3
<210>1
<211>1378
<212>DNA
＜213〉artificial sequence
<400>1
caggcttaac acatgcaagt cgagcgggcg tagcaatacg tcagcggcag acgggtgagt 60
aacgcgtggg aacgtacctt ttggttcgga acaacacagg gaaacttgtg ctaataccgg 120
ataagccctt acggggaaag atttatcgcc gaaagatcgg cccgcgtctg attagctagt 180
tggtgaggta atggctcacc aaggcgacga tcagtagctg gtctgagagg atgatcagcc 240
acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtgggg aatattggac 300
aatgggggca accctgatcc agccatgccg cgtgagtgat gaaggcccta gggttgtaaa 360
gctcttttgt gcgggaagat aatgacggta ccgcaagaat aagccccggc taacttcgtg 420
ccagcagccg cggtaatacg aagggggcta gcgttgctcg gaatcactgg gcgtaaaggg 480
tgcgtaggcg ggtctttaag tcaggggtga aatcctggag ctcaactcca gaactgcctt 540
tgatactgaa gatcttgagt ccgggagagg tgagtggaac tgcgagtgta gaggtgaaat 600
tcgtagatat tcgcaagaac accagtggcg aaggcggctc actggcccgg tactgacgct 660
gaggcacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 720
gatgaatgcc agccgttagt gggtttactc actagtggcg cagctaacgc tttaagcatt 780
ccgcctgggg agtacggtcg caagattaaa actcaaagga attgacgggg gcccgcacaa 840
gcggtggagc atgtggttta attcgacgca acgcgcagaa ccttaccagc ccttgacatg 900
tccaggaccg gtcgcagaga tgtgaccctc tcttcggagc ctggagcaca ggtgctgcat 960
ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccccc 1020
gtccttagtt gctaccattt agttgagcac tctaaggaga ctgccggtga taagccgcga 1080
ggaaggtggg gatgacgtca agtcctcatg gcccttacgg gctgggctac acacgtgcta 1140
caatggcggt gacaatggga tgctaagggg cgacccttcg caaatctcaa aaagccgtct 1200
cagttcggat tgggctctgc aactcgagcc catgaagttg gaatcgctag taatcgtgga 1260
tcagcacgcc acggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg 1320
agttggtttt acctgaagac ggtgcgctaa cccgcaaggg aggcagccgg ccacggta 1378
Claims (9)
1. the knurl bacterial strain RY2 (Bradyrhizobium yuanmingense) of taking root slowly is preserved in Chinese typical culture collection center on November 13rd, 2009, and preserving number is CCTCC NO:M 209266.
2. a method of cultivating the described knurl bacterial strain RY2 of taking root slowly of claim 1 is characterized in that it being streak culture on 28 ± 1 ℃ of following YMA solid mediums.
3. according to the described cultural method of claim 2, what it is characterized in that described YMA solid medium consists of N.F,USP MANNITOL 10g, K
2HPO
40.25g, MgSO
47H
2O 0.2g, KH
2PO
40.25g, NaCl 0.1g, yeast powder 3g, agar 15g.
4. microbial inoculum is characterized in that it being that bacterium liquid mixing thalline sorbent material by the described knurl bacterial strain RY2 of taking root slowly of claim 1 prepares.
5. according to the described microbial inoculum of claim 4, it is characterized in that described thalline sorbent material is the peat composed of rotten mosses, vermiculite or perlite; Blending ratio is 10ml bacterium liquid/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite.
6. the method for the described knurl bacterial strain RY2 of taking root slowly of khuskhus inoculation claim 1, it is characterized in that dresses seed bacterial strain to handle or water with bacterium liquid after shaking bacterium makes microbial inoculum mixed imposing in the soil in the khuskhus sowing or before transplanting in the khuskhus root or with bacterium liquid mixings thalline sorbing material.
7. method according to claim 6, the bacteria containing amount that it is characterized in that described bacterium liquid is 10
9Individual/ml bacterium liquid; Described seed dressing is treated to bacterium liquid soaked seed 45 minutes～50 minutes.
8. the application of the described knurl bacterial strain RY2 of taking root slowly of claim 1 is characterized in that being applied to the intercropping of khuskhus TPRC2.
9. the application of the described knurl bacterial strain RY2 of taking root slowly of claim 1 is characterized in that being applied to the khuskhus of planting in the tieback salt marsh soil.
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| CN105950520B (en) * | 2016-07-18 | 2019-05-10 | 武汉市农业科学技术研究院作物科学研究所 | A kind of rhizobium of efficient phosphate-solubilizing and its application |
| CN106754481B (en) * | 2016-11-26 | 2020-02-07 | 安徽省农业科学院园艺研究所 | Method for screening high-salt-resistant nitrogen-fixing strain from iceberg and related culture medium |
| CN109337847B (en) * | 2018-12-05 | 2020-07-31 | 福建农林大学 | Cassia rhizobium TXR2 and application thereof |
| CN114350559B (en) * | 2021-12-29 | 2023-05-12 | 中国热带农业科学院热带作物品种资源研究所 | Salt-tolerant growth-promoting Liaoning slow rhizobium RY6 strain and application thereof |
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